PEDIATRICS Vol. 107 No. 6 June 2001, pp. 1437-1442

EXPERIENCE AND REASON:
Exposure to Lead Appears to Selectively Alter Metabolism of Cortical Gray Matter

	ABSTRACT

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Objective.  The effects of lead poisoning on the development of children have been examined primarily in the context of behavioral and neuropsychological studies. The purpose of this study was to examine the in vivo use of magnetic resonance spectroscopy (MRS) for the evaluation of the neurotoxic effects of lead on the nervous system. MRS has the ability to monitor brain metabolism by detecting a number of neurochemicals among which is N-acetylaspartate, a metabolite shown to decrease in processes that involve neuronal loss.

Methods.  In the present study we evaluated the metabolism of gray and white matter of frontal cortex using MRS in individuals with elevated blood lead levels and compared the results with those obtained on nonlead-exposed controls.

Results.  Although all of the participants had normal MRI examinations of the brain, the lead-exposed individuals exhibited a significant reduction in the N-acetylaspartate/creatine and phosphocreatine ratios in frontal gray matter compared with the nonlead-exposed controls.

Conclusions.  The findings of this study suggest that lead has an effect on brain metabolites as detected by MRS in vivo. More specifically, we have found statistically significant reduced levels of brain metabolites in gray but not white matter in lead-exposed individuals. These results imply that MRS is able to detect metabolic abnormalities in individuals with lead poisoning.  Key words:  lead, neuronal loss, proton MRS, brain cortex.

During the past several years there has been a growing interest in the effects of exposure to lead on the developing nervous system as well as the mechanisms by which lead disrupts brain function in children. The effects of elevated blood lead levels on the development of children have been examined primarily in the context of behavioral and neuropsychological evaluations, as debate continues on the effects low to moderate lead levels (10-40 µg/dL) have on general behavioral and cognitive functioning. One of the most consistently reported impairments associated with lead exposure at levels as low as 25 µg/dL involves its negative impact on general intellectual functioning.^1-6 A number of centers have conducted longitudinal as well as cross-sectional studies, reporting inverse relations between IQ and dentine or blood lead levels, as well as diminished academic achievement and psychomotor development following low to moderate lead levels.^1-6 Recent studies have been particularly careful at adjusting for confounding variables such as parental intelligence, socioeconomic status, education and home environment, with a general finding that the detrimental effect on IQ remained significant.^6-9 Other neuropsychological impairments reported in conjunction with lead exposure, although less robust, include impaired memory and learning,¹⁰ impaired perceptual integration,¹¹ slower reaction time^12,13 impaired motor development,³ as well as visual-motor integration and serial choice reaction performance.¹⁴

A number of investigators challenged the findings of lowered intellectual and cognitive ability in children, particularly at lower lead levels (10-25 µg/dL), claiming other variables, such as social class, family size and marital relationship,¹⁵ maternal education, quality of care, prenatal and postnatal stressors,¹⁶ and iron deficiency¹⁷ may have caused a detrimental effect on the cognitive functioning of these children. Difficulties in attributing a causal relationship between elevated lead levels and cognitive deterioration also stem from the fact that most studies used epidemiologic and statistical methods to establish the effects of lead, with differences in sample selection, peak and duration of lead levels, differences in measurement of lead levels (blood, dentine, bone) and differences in procedures and statistical methods. Because of these limitations we suggest that there is a strong need for a more objective method with which to evaluate of the effects of lead on the development of children.

Little is known regarding the effects of lead on brain metabolism in vivo, and on structural and functional correlates of brain function. In the human brain, magnetic resonance spectroscopy (MRS) provides a noninvasive risk-free method with which to monitor the biochemistry of acute and chronic stages of disease.^18-20 The development of spatial localized spectroscopic methods that sample the relative levels of mobile metabolites from a volume of tissue defined from a magnetic resonance image has provided a basis for integrating the biochemical information obtained by MRS with the anatomic and pathologic information obtained from magnetic resonance imaging (MRI). This combination of metabolic and anatomic information affords a new means of understanding the origins and time course of progression in variety of diseases. In the brain, MRS has gained widespread acceptance as a method for assessing both neuronal viability as well as demyelination. This acceptance is based on the fact that one of the metabolites identified in proton spectra of the brain, N-acetylaspartate (NAA), is largely confined to neurons,^21,22 and has been proposed as a neuronal marker. In the cortex, NAA is located in neuronal cell bodies whereas in the white matter it is located largely in axons. A decrease in NAA has been proposed as an indicator of neuronal and axonal damage and loss.²³ Proton MRS has been used to study neurodegenerative processes where decreases in NAA have been shown as common findings in patients with Alzheimer's disease,^24,25 Parkinson's disease,²⁶ and Huntington's disease.²⁷ In practice, the decrease in NAA is measured relative to the level of creatine (Cr), a stable metabolite whose level is constant following neuronal loss. As an example, van der Knapp et al²⁸ have demonstrated that increased cerebral atrophy was accompanied by lower ratios of NAA to creatine in patients with demyelination disorders. In children, Kimura et al²⁹ reported abnormally low NAA/Cr ratios in neurologically delayed infants as compared with children with no known developmental delays. Grodd et al³⁰ reported a marked decrease of NAA in children with focal or generalized demyelination. Because there is evidence showing reduced NAA peaks in disease processes involving intellectual deterioration, it is reasonable to hypothesize a decrease in NAA in the brain of children and adults with clinical evidence of lead neurotoxicity. Lopez-Villegas et al³¹ developed a technique to obtain metabolic information differentially from gray and white matter using proton MRS. They reported that spectra from frontal gray matter showed choline-containing compounds (Cho)/Cr and NAA/Cr ratios significantly lower than those from white matter in healthy young adults. They also reported lower Cho and higher Cr content in gray matter. Trope et al³² used this technique to examine a child with elevated blood lead levels, as compared with a first cousin with no lead exposure, as a first step in determining whether this method might serve as a new technique for evaluating the effects lead has on the central nervous system. This study demonstrated that the lead-exposed boy showed a significant alteration in brain metabolites, with a reduction in NAA/Cr ratio for both gray and white matter compared with his nonlead-exposed cousin. Neuropsychological evaluation demonstrated areas of impairment in the lead-exposed child, including difficulties in academic skills of reading, writing and arithmetic, deficient linguistic skills and attention. By contrast, neuropsychological examination of the cousin was within normal limits. As a next step, we have used the methods described previously by Trope et al³² and Lopez-Villegas et al³¹ in the present study to examine proton spectra obtained from individuals with elevated lead levels and compare them to spectra obtained from healthy, nonexposed individuals.

	METHODS

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Subjects

Sixteen individuals with documented blood lead levels ranging from 23 to 65 µg/dL (mean lead = 39.93 µg/dL, standard deviation [SD] = 13.39) were included in the subject group. There were 5 boys and 11 girls. The mean age at the time of testing was 8 years, 9 months (range: 4-21 years, SD = 4.36). All of the individuals in the subject group came to medical attention before the age of 5 years (range: 10-60 months, mean = 27.68 months, SD = 13.82). The control group consisted of 5 individuals (siblings/cousins of Subject Group) whose reported lead levels as indicated in their pediatrician records never exceeded 10 µg/dL. There were 3 boys and 2 girls. The mean age at the time of testing was 8 years, 6 months (range: 6-11 years, SD = 1.62). All of the individuals were evaluated using the MRI and MRS methods at the University of Pennsylvania Medical Center. The only significant difference between the 2 groups is the exposure to lead in the subject but not the control group. None of the participants was known to have neurologic deficits.

Table 1 summarizes subject characteristics for the two groups.

Procedures

Informed consent was obtained from the parents/guardians of all participants. All of the magnetic resonance studies were performed at the Hospital of the University of Pennsylvania in Philadelphia, on a 1.5T Signa system (GE Medical Systems, Milwaukee, WI). MRI was performed with a standard quadrature head coil. The children were not sedated and participated willingly.

After conventional MRI, the standard quadrature head coil was replaced by a 3-inch surface coil that was positioned over the left frontal region immediately supraciliary. A sagittal localizer was obtained followed by axial 3-dimensional spoiled gradient acquired in the steady state (GRASS) (3D-SPGR) images (256 × 256 matrix, 8-cm field of view, repetition time [TR] 22.4 ms, echo time [TE] 7.5 ms, 45 flip angle, 2 acquisitions, 1.5-mm thickness and 28 slices). The 3D-SPGR images provide high contrast between gray and white matter and were used to choose the voxel of interest (VOI) for the spectroscopic study. Immediately after high resolution MRI, 1-dimensional (1D)-proton spectra were obtained with the stimulated-echo acquisition mode for localization. Water suppression was achieved by using 3 chemical shift-selective radiofrequency pulses followed by a dephasing gradient applied on each of the 3 axes. The sequence parameters included the following: 19-cm field of view, spectral bandwidth 2500 Hz, 32 phase-encoding steps, TR 2000 ms, TE 31 ms, mixing time 10.6 ms, 2048 complex points, 8-step phase cycling and 16 acquisitions. We selected a VOI of 30-40 × 6 × 10 mm including cortical gray and white matter. Spectra from contiguous 6 × 6 × 10-mm voxels were obtained from the VOI by 1D phase-encoding. Cortical sulci were included in the VOI in all cases. Because the thickness of cortical gray matter is about 3 mm³³ the inclusion of cortical sulci in the VOI guarantee about 6 mm-thickness of gray matter. To avoid partial volume effects, the spatial distribution of gray and white matter included in the VOI has been checked to be relatively invariant in at least 6 of the MR images (1.5 mm-contiguous slices) that contributed to the MRS slice (10 mm-thickness). Scalp and marrow were excluded from the VOI to prevent contamination from lipids. Gradient shimming on the VOI and optimization of solvent suppression were performed before the start of the acquisition. The spectral acquisition time was 17 minutes and the total examination time, including MRI and MRS studies, was ~55 minutes. The MR procedure was well-tolerated by all subjects.

The spectral processing was performed with ProNMR (Softpulse Software, Guelph, Ontario, Canada) using zero filling to 4K data points, 1.5 Hz line broadening applied in the time domain, 2-dimensional Fourier transformation, and zero-order phase. Areas under the peaks were determined using a Marquardt fitting routine to Lorentzian line shapes in the frequency domain and peak area ratios were calculated. MRI and MRS were evaluated blind to the status of the subjects. The MRI films were reviewed by an attending radiologist at the Department of Radiology of the University of Pennsylvania.

The peak assignments were made based on the published literature and the chemical shifts were determined using NAA as a chemical shift standard. The following resonances were assigned: NAA, 2.0 ppm, 2.6 ppm; Cr, 3.0 ppm, 3.9 ppm; Cho, 3.2 ppm; and myoinositol (mI), 3.5 ppm. The region between 2.1 and 2.5 ppm contains peaks from glutamate, glutamine, gamma-amino butyric acid and NAA. These peaks could not be resolved because of the overlap of resonances. Other peaks from glutamate and glutamine are contained in the region between 3.6 and 3.8 ppm. Residual lipid signals were identified in the region between 0.5 and 1.5 ppm. The peak at 2.01 ppm and 3.0 ppm were used for the quantification of NAA and Cr, respectively.

The mean and SD for the mean values determined from each individual in each of the 2 groups were calculated for each metabolite ratio. Statistical comparisons between metabolite ratios from gray and white matter were made using 2-tailed unpaired Student's t test for each of the metabolite shifts using Statview Version 5.01 (SAS Institute, Cary, NC).

	RESULTS

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All of the MRI examinations were reported normal with no evidence of structural abnormalities for any of the participants.

A representative study showing the VOI prescription in the left prefrontal lobe along with the stack-plot of proton spectra from adjacent voxels obtained by 1D phase-encoding is shown in Fig 1.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   A representative study showing the VOI prescription in the left prefrontal lobe along with the stack-plot of proton spectra from adjacent voxels obtained by 1D phase-encoding.

The signal-to-noise ratio from spectra coming from the margins of the VOI was lower compared with intermediate voxels probably resulting from partial volume effects. Typical individual spectra from frontal gray matter and white matter with the principal metabolites identified are shown in Fig 2 for the subject group and the control group.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   A, Spectra from frontal gray matter with the principal metabolites identified for a subject (top) with high blood lead levels and normal control (bottom). B, Spectra from frontal white matter with the principal metabolites identified for a subject with high blood lead levels (top) and normal control (bottom).

The results of an analysis of peak area ratios for gray and white matter are summarized in Table 2 for the 2 groups.

The NAA/Cr ratio in gray matter was significantly lower for the subject group as compared with the control group (unpaired t test, P = .0345). In contrast, none of the other ratios in gray matter or the ratios obtained in white matter (see Table 2) was statistically significantly different.

Figure 3 shows a plot of the variation in the NAA/Cr ratio in gray matter with the highest lead level in the blood of each individual in the subject group. The best-fit linear regression showed an r value of 0.05 indicating that there is no correlation between these 2 variables.

View larger version (9K): [in this window] [in a new window]   Fig. 3.   A plot of the variation in the NAA/Cr ratio in gray matter with the highest lead level in the blood of each individual in the Subject group.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion Conclusion References

The present study examined in vivo metabolic differences in the brains of individuals exposed to lead as compared with healthy controls. The individuals who participated in this study and included in the control group were relatives (siblings, cousins) of the individuals comprising the subject group. Both subjects and controls in this study came from the same socioeconomic and home environments, thus eliminating a number of variables usually regarded as factors in behavioral lead studies. The only significant difference between them was elevated lead levels in the subject but not in the control group. The MRS study of the healthy, nonlead-exposed individuals resulted in spectra entirely consistent with the spectral pattern reported in previous studies for healthy individuals^31,34,35 documenting the levels of these metabolites in the normal adult brain, as well as the estimated metabolite concentrations. These studies demonstrated that spectra from frontal gray matter are characterized by a lower Cho/Cr ratio and NAA/Cr ratio as compared with those obtained from white matter. Using the same technique as used in the present study, Lopez-Villegas et al³¹ also reported that in healthy young adults, there were no differences in mI/Cr ratios for gray and white matter. The spectra obtained from individuals in the control group showed the same pattern of metabolite ratios. It is appropriate to compare the spectra obtained from the children who participated in the present study with those obtained in the study of Lopez-Villegas et al of young adults, because it has been reported that the levels of metabolites in the brains of children reach their adult levels by the age of 3 years.²⁹

In contrast to the spectra obtained from the control group, the spectra obtained from the lead-exposed individuals showed lower NAA/Cr ratios for gray matter. Previous studies have linked lowered NAA/Cr ratios to neuronal loss and decline in intellectual functioning.^21,24,28,29 Therefore, the lowered NAA/Cr ratio in the lead-exposed individuals is suggestive of neuronal loss in the region examined. There is no indication in any of the individuals examined of any developmental history for any event other than his/her lead exposure. It is therefore possible that the reduction in NAA/Cr ratio may be a direct result of his/her elevated lead levels. Alternatively, in the absence of premorbid MRS measurements, the loss in NAA/Cr in these subjects may reflect a selective vulnerability to lead exposure rather than a direct effect of lead neurotoxicity.

It is of note that we did not find a significant correlation between the highest reported blood lead level and the NAA/Cr ratio in frontal gray matter. The reason for this is unclear. We acknowledge the possibility that the highest blood lead level reported in this study may not reflect the highest exposure to lead. It is possible that the NAA/Cr ratio may reflect other aspects of lead exposure such as the duration of exposure or time since the highest level of exposure. These possibilities could be tested in a cohort with more detailed lead exposure data.

The relationship between NAA/Cr and neuronal viability was demonstrated by Cheng and coworkers.³⁶ This group found an inverse linear correlation between surviving pyramidal neurons per unit area on microscopic examination with the NAA/Cr ratio determined by MRS in brain samples obtained from patients with Pick disease. To our knowledge, this is the first study to demonstrate a histopathological validation of MRS decreases in NAA.

In our previous case study, we found that the NAA/Cr ratio was lower in white matter as well as gray matter. In the larger group, there was a trend for a lower NAA/Cr ratio in white matter in the lead-exposed group, which did not reach statistical significance. It is possible that this trend might become significant when a larger cohort is studied. We were able to obtain high quality spectra from voxels as small as 0.36 cm³ at 1.5T. The spatial resolution used in the present study is sufficient to obtain spectra from voxels almost exclusively comprising gray matter. The 1D phase-encoding approach used has the advantage of obtaining several spectra simultaneously in a relatively short period of time. The present study has allowed us to examine the spectroscopic patterns of frontal gray and white matter after lead exposure relative to the normal pattern seen in healthy children and adults. This provides opportunities for the investigation of the brain of children and adults with lead poisoning to determine more precisely the effects of lead on the brain, and to examine any regional metabolic abnormalities.

We have demonstrated differences in metabolites in regions in the frontal lobe, which are particularly relevant, as the signature effects of lead neurotoxicity involve functions of the prefrontal and frontal lobes such as attention and executive functions, social-behavioral conduct, and impulse control. Additional studies confirming these differences as well as sampling different regions in the brain will be helpful in establishing whether lead affects specific brain regions or, alternatively, affects the brain more diffusely. The potential for this technique in determining the specific effects of lead on the central nervous system appears feasible and significant.

	CONCLUSION

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Both subjects and controls in this study came from the same socioeconomic and home environments, thus eliminating a number of variables usually regarded as cofounders in behavioral lead studies. Although MRI examinations were normal for both groups, MRS metabolites in the lead-exposed subjects were significantly reduced as compared with the controls, suggesting interference with neuronal functioning after lead exposure. More specifically, this study showed that individuals with a history of moderate elevations in blood lead levels show reduced NAA/Cr ratios in frontal gray matter. It was demonstrated that MRS can be used as a technique to measure brain metabolites in vivo, and provides us with a measure of neuronal viability. This finding is important in relation to children exposed to lead, as it enables us to evaluate the degree of neuronal loss.

Idit Trope, PhD
Dolores Lopez-Villegas, MD^*
Kim M. Cecil, PhD^*
Robert E. Lenkinski, PhD^*
Department of Psychiatry and ^* Department of Radiology
University of Pennsylvania Medical Center
Philadelphia, PA 19104

	FOOTNOTES

Received for publication Apr 4, 2000; accepted Sep 21, 2000.

Reprint requests to (I.T.) 818-822 Pine St, Suite 1E, Philadelphia, PA 19107. E-mail: idit{at}mindspring.com

	ABBREVIATIONS

MRS, magnetic resonance spectroscopy; MRI, magnetic resonance imaging; NAA, N-acetylaspartate; Cr, creatine and phosphocreatine; Cho, choline-containing compounds; 3DSPGR, 3-dimensional spoiled GRASS, gradient acquired in the steady state; TR, repetition time; TE, echo time; VOI, voxel of interest; 1D, 1-dimensional; mI, myoinositol.

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